Among communicable diseases, tuberculosis is the second leading cause of death worldwide. New antimycobacterial drugs that allow a shortened but effective chemotherapy, that target replicating as well as non-replicating mycobacteria, and are active against multidrug-resistant Mycobacterium tuberculosis (Mtb) are urgently needed. Recent advances in functional genomics identified a number of essential genes that represent potential targets for the development of novel antibiotics. However, essential genes are difficult to study and it is unknown which of them are required during infection, in particular during the chronic phase of tuberculosis. To address this, we propose to develop mycobacterial tet-on and tet-off systems that allow to regulate gene expression of Mtb in vitro and in vivo. In Aim I we will optimize tet-on and develop tet-off systems for efficient gene induction and silencing. In Aim II we will use these systems to generate conditional knockout strains and analyze the role of glycolysis and gluconeogenesis during growth of Mtb with defined carbon sources. In Aim III we will use tet systems to regulate gene expression in Mtb during growth in mice and determine if glycolysis and gluconeogenesis are required for in vivo growth of Mtb. The experiments proposed here will (i) provide the TB-research community with a molecular tool that allows expression of mycobacterial genes at different time points during an infection (ii)provide a molecular tool for the validation of new drug targets; and (iii) provide insights into the metabolic adaptations required for Mtb to grow within its host. Many of the experiments focus on generally conserved genes and will, therefore, lead to insights that may be relevant to other pathogens and diseases. Lay Summary: Tuberculosis (TB) is one of the world's most devastating diseases. It is responsible for more than two million deaths and eight million new cases annually. Work outlined in this proposal will develop molecular tools to aid the understanding of how Mycobacterium tuberculosis grows and persists within its host. It will help identify and validate novel drug targets that might lead to the development of antibiotics against replicating and non-replicating Mycobacterium tuberculosis and thus shorten anti-tuberculosis drug therapy.